WO2023223354A1 - Novel mutations for determining drug resistance in tuberculosis treatment and implementations thereof - Google Patents
Novel mutations for determining drug resistance in tuberculosis treatment and implementations thereof Download PDFInfo
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- WO2023223354A1 WO2023223354A1 PCT/IN2023/050468 IN2023050468W WO2023223354A1 WO 2023223354 A1 WO2023223354 A1 WO 2023223354A1 IN 2023050468 W IN2023050468 W IN 2023050468W WO 2023223354 A1 WO2023223354 A1 WO 2023223354A1
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
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- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
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- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
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- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The present disclosure provides a polynucleotide fragment of M. tuberculosis having a nucleic acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16. Each of the disclosed polynucleotide has a nucleotide substitution, which indicates resistance to antibiotic for treatment of tuberculosis. The disclosure further provides a method for determining whether a subject infected with M. tuberculosis is resistant or susceptible to a specific antibiotic for the treatment of tuberculosis using the disclosed nucleotide substitutions.
Description
NOVEL MUTATIONS FOR DETERMINING DRUG RESISTANCE IN TUBERCULOSIS TREATMENT AND IMPLEMENTATIONS THEREOF
FIELD OF INVENTION
[001] The present disclosure broadly relates to the detection of drug resistance in subjects suffering from tuberculosis. Particularly, the present disclosure discloses polynucleotide fragments consisting of nucleotide substitutions for determining if a subject infected with M. tuberculosis is resistant or susceptible to an antibiotic.
BACKGROUND OF INVENTION
[002] Tuberculosis (TB) is a communicable disease, which primarily affects the lungs. TB is caused by the bacterium, Mycobacterium tuberculosis (Mtb) and is one of the leading causes of mortality worldwide. According to the World Health Organization Report (Global tuberculosis report 2021. Geneva: World Health Organization; 2021. Licence: CC BY -NCSA 3.0 IGO), an estimated 9.9 million people were infected and 1.5 million died of TB in 2020. TB is curable, with the current treatment regimen including the four first line drugs- isoniazid, rifampicin, ethambutol, and pyrazinamide. However, severe side effects from these drugs; long treatment regimens leading to noncompliance by TB patients have led to drug resistant TB, which is a serious public health crisis. Resistance to at least isoniazid and rifampicin, the two most effective first line drug is referred to as multi-drug resistant TB (MDR-TB) (WHO consolidated guidelines on tuberculosis. Module 4: treatment - drug-resistant tuberculosis treatment. Geneva: World Health Organization; 2020. Licence: CC BY-NC-SA 3.0 IGO) and is curable by fluoroquinolones, but these are toxic, expensive and require even longer treatment schedules. Thus, early detection of MDR-TB, followed with appropriate therapy is essential in preventing the development of other detrimental forms of drug resistant TB.
[003] The standard method for detecting drug resistance involves culturing the bacteria in the presence and absence of the drug. The culture -based drug susceptibility testing is unreliable due to the slow growth rate of Mtb (typically 4-6 weeks) and poor reproducibility of the test results. As drug resistance may be caused
due to genetic variations such as mutations in the nucleotide sequence of the bacterium, various high-throughput sequencing and genotyping approaches have been developed to identify these resistance causing mutations (lame Emane AK, Guo X, Takiff HE, Liu S. Drug resistance, fitness, and compensatory mutations in Mycobacterium tuberculosis. Tuberculosis (Edinb). 2021 Jul;129:102091. doi: 10.1016/j.tube.2021.102091. Epub 2021 May 21). However, as may be understood, drug resistance may not occur solely owing to such genetic variations. In certain instances, such genetic variations may be compensated for by some other genetic factor, owing to which the strain under consideration may still be susceptible, despite the presence of the genetic variations. Furthermore, on detecting a new genetic variation in a strain of the pathogen, conventional approaches may not provide any drug recommendation.
[004] US7919234B2 provides methods and compositions for the detection of disease caused by infectious agents and microbes. In particular, methods and compositions comprising novel combinations of nucleic acid amplification and drug susceptibility technologies are provided.
[005] US 10982291B2 relates generally to methods of detecting, diagnosing, and/or identifying pathogens, e.g., infectious disease pathogens and determining their drug sensitivity and appropriate methods of treatment. The disclosure also relates generally to methods of monitoring pathogen infection in individual subjects as well as larger populations of subjects.
[006] However, the methods known in the art suffer from certain drawbacks as discussed hereinabove, therefore, there is an immediate need to devise a quick and accurate approach to identify the drug resistance status in a target strain based on genetic variations and to further guide the appropriate course of treatment at the earliest.
SUMMARY OF THE INVENTION
[007] In an aspect of the present disclosure, there is provided a polynucleotide fragment of M. tuberculosis having a nucleic acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID
NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16; wherein: SEQ ID NO: 1 comprises a nucleotide substitution at C216T; SEQ ID NO: 2 comprises a nucleotide substitution at G705A; SEQ ID NO: 3 comprises a nucleotide substitution at A174C; SEQ ID NO: 4 comprises a nucleotide substitution at G1177C; SEQ ID NO: 5 comprises a nucleotide substitution at C674G; SEQ ID NO: 6 comprises a nucleotide substitution at C771T; SEQ ID NO: 7 comprises a nucleotide substitution at C343T; SEQ ID NO: 8 comprises a nucleotide substitution at G466T; SEQ ID NO: 9 comprises a nucleotide substitution at CCGA[2522]C; SEQ ID NO: 10 comprises a nucleotide substitution at T533G; SEQ ID NO: 11 comprises a nucleotide substitution atC112T; SEQ ID NO: 12 comprises a nucleotide substitution at G355A; SEQ ID NO: 13 comprises a nucleotide substitution at C383A; SEQ ID NO: 14 comprises a nucleotide substitution at T30C; SEQ ID NO: 15 comprises a nucleotide substitution at C643T; and SEQ ID NO: 16 comprises a nucleotide substitution at T633C; wherein, the nucleotide substitution indicates resistance to antibiotic for treatment of tuberculosis.
[008] In an aspect of the present disclosure, there is provided a method for determining resistance or susceptibility of a subject to a specific antibiotic for the treatment of tuberculosis, the method comprising the steps of: (a) obtaining a nucleic acid from a sample of the subject; and (b) sequencing the nucleic acid to detect the presence or absence of the substitutions described herein; wherein, the presence of the substitution in the nucleic acid determines that the subject is resistant to the specific antibiotic.
[009] In an aspect of the present disclosure, there is provided a method for determining resistance or susceptibility of a subject to a specific antibiotic for treatment of tuberculosis, the method comprising the steps of: (a) obtaining a nucleic acid from a sample of the subject; and (b) performing a PCR amplification with primer sets to obtain an amplicon and sequencing the amplicon to detect for the presence or absence of the substitutions as described herein, wherein the primer set comprises primers having a sequence selected from sequence as set forth in: SEQ ID NO: 17 and SEQ ID NO: 18 for detecting mutation in SEQ ID NO: 1; SEQ ID NO:
21 and SEQ ID NO: 22 for detecting mutation in SEQ ID NO: 2; SEQ ID NO: 25 and SEQ ID NO: 26 for detecting mutation in SEQ ID NO: 3; SEQ ID NO: 29 and SEQ ID NO: 30 for detecting mutation in SEQ ID NO: 4; SEQ ID NO: 33 and SEQ ID NO: 34 for detecting mutation in SEQ ID NO: 5; SEQ ID NO: 37 and SEQ ID NO: 38 for detecting mutation in SEQ ID NO: 6; SEQ ID NO: 41 and SEQ ID NO: 42 for detecting mutation in SEQ ID NO: 7; SEQ ID NO: 45 and SEQ ID NO: 46 for detecting mutation in SEQ ID NO: 8; SEQ ID NO: 49 and SEQ ID NO: 50 for detecting mutation in SEQ ID NO: 9; SEQ ID NO: 53 and SEQ ID NO: 54 for detecting mutation in SEQ ID NO: 10; SEQ ID NO: 57 and SEQ ID NO: 58 for detecting mutation in SEQ ID NO: 11; SEQ ID NO: 61 and SEQ ID NO: 62 for detecting mutation in SEQ ID NO: 12; SEQ ID NO: 65 and SEQ ID NO: 66 for detecting mutation in SEQ ID NO: 13; SEQ ID NO: 69 and SEQ ID NO: 70 for detecting mutation in SEQ ID NO: 14; SEQ ID NO: 73 and SEQ ID NO: 74 for detecting mutation in SEQ ID NO: 15; SEQ ID NO: 77 and SEQ ID NO: 78 for detecting mutation in SEQ ID NO: 16; or combinations thereof; wherein, the presence of the substitution in the amplicon indicates the resistance to a specific antibiotic.
[0010] In an aspect of the present disclosure, there is provided a method of preparing a treatment regimen for a subject infected with M. tuberculosis, the method comprising the steps of: (a) determining whether the subject infected with tuberculosis is resistant to an antibiotic using the method as described herein; and (b) selecting an antibiotic for which the subject having tuberculosis is not resistant as determined in step (a) for preparing a treatment regimen, wherein the antibiotic is selected from the group consisting of isoniazid, rifampicin/rifampin, ethambutol, pyrazinamide, rifabutin, rifapentine, streptomycin, moxifloxacin, ciprofloxacin, levofloxacin, ofloxacin, p-aminosalicylic acid, ethionamide, prothionamide, cycloserine, terizidone, capreomycin, kanamycin, amikacin, linezolid, delamanid, pretomanid, clofazimine and combinations thereof.
[0011] In an aspect of the present disclosure, there is provided a kit for determining resistance or susceptibility of a subject to a specific antibiotic for the treatment of tuberculosis, comprising one or more of primer set; wherein the primer set comprises
primers having a sequence as set forth in: SEQ ID NO: 17 and SEQ ID NO: 18 for detecting mutation in SEQ ID NO: 1; SEQ ID NO: 21 and SEQ ID NO: 22 for detecting mutation in SEQ ID NO: 2; SEQ ID NO: 25 and SEQ ID NO: 26 for detecting mutation in SEQ ID NO: 3; SEQ ID NO: 29 and SEQ ID NO: 30 for detecting mutation in SEQ ID NO: 4; SEQ ID NO: 33 and SEQ ID NO: 34 for detecting mutation in SEQ ID NO: 5; SEQ ID NO: 37 and SEQ ID NO: 38 for detecting mutation in SEQ ID NO: 6; SEQ ID NO: 41 and SEQ ID NO: 42 for detecting mutation in SEQ ID NO: 7; SEQ ID NO: 45 and SEQ ID NO: 46 for detecting mutation in SEQ ID NO: 8; SEQ ID NO: 49 and SEQ ID NO: 50 for detecting mutation in SEQ ID NO: 9; SEQ ID NO: 53 and SEQ ID NO: 54 for detecting mutation in SEQ ID NO: 1; SEQ ID NO: 57 and SEQ ID NO: 58 for detecting mutation in SEQ ID NO: 11; SEQ ID NO: 61 and SEQ ID NO: 62 for detecting mutation in SEQ ID NO: 12; SEQ ID NO: 65 and SEQ ID NO: 66 for detecting mutation in SEQ ID NO: 13; SEQ ID NO: 69 and SEQ ID NO: 70 for detecting mutation in SEQ ID NO: 14; SEQ ID NO: 73 and SEQ ID NO: 74 for detecting mutation in SEQ ID NO: 15; or SEQ ID NO: 77 or SEQ ID NO: 78 for detecting mutation in SEQ ID NO: 16.
[0012] In an aspect of the present disclosure, there is provided a panel of nucleotide fragments for determining antibiotic resistance in a subject comprising at least one polynucleotide fragment of M. tuberculosis, having a nucleic acid sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15 or SEQ ID NO: 16, wherein: SEQ ID NO: 1 comprises a nucleotide substitution at C216T; SEQ ID NO: 2 has a nucleotide substitution at G705A; SEQ ID NO: 3 comprises a nucleotide substitution at A174C; SEQ ID NO: 4 comprises a nucleotide substitution at G1177C; SEQ ID NO: 5 comprises a nucleotide substitution at C674G; SEQ ID NO: 6 comprises a nucleotide substitution at C771T; SEQ ID NO: 7 comprises a nucleotide substitution at C343T; SEQ ID NO: 8 comprises a nucleotide substitution at G466T; SEQ ID NO: 9 comprises a nucleotide substitution at CCGA[2522]C; SEQ ID NO: 10 comprises a nucleotide substitution at T533G;
SEQ ID NO: 11 comprises a nucleotide substitution at C112T; SEQ ID NO: 12 comprises a nucleotide substitution at G355A; SEQ ID NO: 13 comprises a nucleotide substitution at C383A; SEQ ID NO: 14 comprises a nucleotide substitution at T30C; SEQ ID NO: 15 comprises a nucleotide substitution at C643T; and SEQ ID NO: 16 comprises a nucleotide substitution at T633T; wherein, the presence of nucleotide substitution indicates resistance to antibiotic for treatment of tuberculosis.
[0013] These and other features, aspects, and advantages of the present subject matter will be better understood with reference to the following description and appended claims. This summary is provided to introduce a selection of concepts in a simplified form. This summary is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used to limit the scope of the claimed subject matter.
BRIEF DESCRIPTION OF DRAWINGS
[0014] The following drawings form a part of the present specification and are included to further illustrate aspects of the present disclosure. The disclosure may be better understood by reference to the drawings in combination with the detailed description of the specific embodiments presented herein.
[0015] Figure 1 is a schematic representation of the workflow for identifying the disclosed novel mutations, in accordance with an embodiment of the present disclosure.
[0016] Figure 2 is a schematic representation of the criteria for filtering and identifying the disclosed novel mutations, in accordance with an embodiment of the present disclosure.
DETAILED DESCRIPTION OF THE INVENTION
[0017] Those skilled in the art will be aware that the present disclosure is subject to variations and modifications other than those specifically described. It is to be understood that the present disclosure includes all such variations and modifications. The disclosure also includes all such steps, features, compositions, and compounds
referred to or indicated in this specification, individually or collectively, and any and all combinations of any or more of such steps or features.
[0018] Sequences used in the present disclosure:
[0019] SEQ ID NO: 1 depicts the nucleotide sequence of moeY gene.
[0020] NCBI Gene ID: 886839
[0021] SEQ ID NO: 2 depicts the nucleotide sequence of Rv2957 gene.
[0022] NCBI Gene ID: 887258
[0023] SEQ ID NO: 3 depicts the nucleotide sequence of Rv0177 gene.
[0024] NCBI Gene ID: 886795
[0025] SEQ ID NO: 4 depicts the nucleotide sequence of Rv3727 gene.
[0026] NCBI Gene ID: 885766
[0027] SEQ ID NO: 5 depicts the nucleotide sequence of Rv2522c gene.
[0028] NCBI Gene ID: 887375
[0029] SEQ ID NO: 6 depicts the nucleotide sequence of Rv0260c gene.
[0030] NCBI Gene ID: 886651
[0031] SEQ ID NO: 7 depicts the nucleotide sequence of Rv3770c gene.
[0032] NCBI Gene ID: 886104
[0033] SEQ ID NO: 8 depicts the nucleotide sequence of Rv2743c gene.
[0034] NCBI Gene ID: 887779
[0035] SEQ ID NO: 9 depicts the nucleotide sequence of Rvl358 gene.
[0036] NCBI Gene ID: 886817
[0037] SEQ ID NO: 10 depicts the nucleotide sequence of Rv0845 gene.
[0038] NCBI Gene ID: 885218
[0039] SEQ ID NO: 11 depicts the nucleotide sequence of Rv3603c gene.
[0040] NCBI Gene ID: 885583
[0041] SEQ ID NO: 12 depicts the nucleotide sequence of argS gene.
[0042] NCBI Gene ID: 886964
[0043] SEQ ID NO: 13 depicts the nucleotide sequence of kdpD gene.
[0044] NCBI Gene ID: 886084
[0045] SEQ ID NO: 14 depicts the nucleotide sequence of esxW gene.
[0046] NCBI Gene ID: 885787
[0047] SEQ ID NO: 15 depicts the nucleotide sequence of Rvl520 gene.
[0048] NCBI Gene ID: 886447
[0049] SEQ ID NO: 16 depicts the nucleotide sequence of IppB gene.
[0050] NCBI Gene ID: 888054
[0051] SEQ ID NO: 17 depicts the nucleotide sequence of forward primer for moeY.
[0052] 5’-GTGACCATCCCACACGAGGG -3’
[0053] SEQ ID NO: 18 depicts the nucleotide sequence of reverse primer for moeY.
[0054] 5’- CTATCTGTGCGGCTCTGGTA -3’
[0055] SEQ ID NO: 19 depicts the nucleotide sequence of forward primer for moeY.
[0056] 5'- CGGTGATGAGGTGCCTGTT -3'
[0057] SEQ ID NO: 20 depicts the nucleotide sequence of reverse primer for moeY.
[0058] 5' - GCGAAGCGATGGGCCTATTA -3'
[0059] SEQ ID NO: 21 depicts the nucleotide sequence of forward primer for Rv2957.
[0060] 5'- ATGGTGCAGACGAAACGATA -3'
[0061] SEQ ID NO: 22 depicts the nucleotide sequence of reverse primer for Rv2957.
[0062] 5'- CTAACGTCGGCGCCGCCAAG -3'
[0063] SEQ ID NO: 23 depicts the nucleotide sequence of forward primer for Rv2957.
[0064] 5'- ACGATCGTCGACAAGGAGTT -3'
[0065] SEQ ID NO: 24 depicts the nucleotide sequence of reverse primer for Rv2957.
[0066] 5'- CCTGCGCACCAGAACTATGA -3'
[0067] SEQ ID NO: 25 depicts the nucleotide sequence of forward primer for Rv0177.
[0068] 5’ - ATGAGCCCCCGTCGTAAGTT-3’
[0069] SEQ ID NO: 26 depicts the nucleotide sequence of reverse primer for Rv0177.
[0070] 5’- TCAGATCACCGGAAGCAGAC-3’
[0071] SEQ ID NO: 27 depicts the nucleotide sequence of forward primer for Rv0177.
[0072] 5’- CGGATCTCCCATGAATCGCA-3’
[0073] SEQ ID NO: 28 depicts the nucleotide sequence of reverse primer for Rv0177.
[0074] 5’- GGATCCGGTGACGTGAACAT -3’
[0075] SEQ ID NO: 29 depicts the nucleotide sequence of forward primer for Rv3727.
[0076] 5'- ATGAAACCGTCGCCTGCCGA -3'
[0077] SEQ ID NO: 30 depicts the nucleotide sequence of reverse primer for Rv3727.
[0078] 5'- CTAACTTGCAGAGCGATCCA-3'
[0079] SEQ ID NO: 31 depicts the nucleotide sequence of forward primer for Rv3727.
[0080] 5'- TTCTGGAAAAACGTCCGCCT -3'
[0081] SEQ ID NO: 32 depicts the nucleotide sequence of reverse primer for Rv3727.
[0082] 5'- GCCGATCAAAAACAGGTCCG -3'
[0083] SEQ ID NO: 33 depicts the nucleotide sequence of forward primer for Rv2522c.
[0084] 5'- CAGTACAACGTCAAGGCCAC -3'
[0085] SEQ ID NO: 34 depicts the nucleotide sequence of reverse primer for Rv2522c.
[0086] 5'- GACCCACGAAGGGGATACCG- 3'
[0087] SEQ ID NO: 35 depicts the nucleotide sequence of forward primer for Rv2522c.
[0088] 5’- CCAACAGCCGCACTAGCA-3’
[0089] SEQ ID NO: 36 depicts the nucleotide sequence of reverse primer for Rv2522c.
[0090] 5’- GCTTTGACGGTGTCGCTACG -3’
[0091] SEQ ID NO: 37 depicts the nucleotide sequence of forward primer for Rv0260c.
[0092] 5’- ATGGCCCAGGCACACTCGGC -3’
[0093] SEQ ID NO: 38 depicts the nucleotide sequence of reverse primer for Rv0260c.
[0094] 5’- TCATACGTCATCGTGCCGGC-3’
[0095] SEQ ID NO: 39 depicts the nucleotide sequence of forward primer for Rv0260c.
[0096] 5’- CTTTGAACGTGCACGAACCC -3’
[0097] SEQ ID NO: 40 depicts the nucleotide sequence of reverse primer for Rv0260c.
[0098] 5’- CGTTGATCCGAAAGGGCGTC -3’
[0099] SEQ ID NO: 41 depicts the nucleotide sequence of forward primer for Rv3770c.
[00100] 5’- ATGCTTAGTGGCATACAACA-3’
[00101] SEQ ID NO: 42 depicts the nucleotide sequence of reverse primer for Rv3770c.
[00102] 5 ’ -TTACCTGGCGGTGAGCGCGA-3 ’
[00103] SEQ ID NO: 43 depicts the nucleotide sequence of forward primer for Rv3770c.
[00104] 5’- TGAGCTGTCCCCAGAGCA -3’
[00105] SEQ ID NO: 44 depicts the nucleotide sequence of reverse primer for Rv3770c.
[00106] 5’ -GATGGTCCCCTCCTGACCA-3’
[00107] SEQ ID NO: 45 depicts the nucleotide sequence of forward primer for Rv2743c.
[00108] 5’ -ATGGCAGTGAAAGCGGGTCA -3’
[00109] SEQ ID NO: 46 depicts the nucleotide sequence of reverse primer for Rv2743c.
[00110] 5’ -CTAACGCCGGGGCAATCCGC -3’
[00111] SEQ ID NO: 47 depicts the nucleotide sequence of forward primer for Rv2743c.
[00112] 5’ CGTTAGGTCGCGGATCTCGTC -3’
[00113] SEQ ID NO: 48 depicts the nucleotide sequence of reverse primer for Rv2743c.
[00114] 5-TCCGAACGCGGGTTCTTCTC’-3’SEQ ID NO: 49 depicts the nucleotide sequence of forward primer for Rvl358.
[00115] 5’-ATGTTCTTGTCCGCACCGGC -3’
[00116] SEQ ID NO: 50 depicts the nucleotide sequence of reverse primer for Rvl358.
[00117] 5 -CTAGGTCGAGTGTTGCGCGG’-3’
[00118] SEQ ID NO: 51 depicts the nucleotide sequence of forward primer for Rvl358.
[00119] 5’ -CGAACGGAGCATTGGCACTA-3’
[00120] SEQ ID NO: 52 depicts the nucleotide sequence of reverse primer for Rvl358.
[00121] 5’ - TGCCATGGCTTCGACATACA -3’
[00122] SEQ ID NO: 53 depicts the nucleotide sequence of forward primer for Rv0845.
[00123] 5’ - GTGCCCAGCTACGGCAACCT -3’
[00124] SEQ ID NO: 54 depicts the nucleotide sequence of reverse primer for Rv0845.
[00125] 5’ TCACCGTTTCAGTGGTAGTT -3’
[00126] SEQ ID NO: 55 depicts the nucleotide sequence of forward primer for Rv0845.
[00127] 5’- GCCTGTACGCTAGTCGGATT 3’
[00128] SEQ ID NO: 56 depicts the nucleotide sequence of reverse primer for Rv0845.
[00129] 5’ CATAGAGCGCGAACCGAAAT -3’
[00130] SEQ ID NO: 57 depicts the nucleotide sequence of forward primer for Rv3603c.
[00131] 5 ’ - ATGGAGCGGTTCGACGGTTT-3 ’
[00132] SEQ ID NO: 58 depicts the nucleotide sequence of reverse primer for Rv3603c.
[00133] 5 ’ -TC ATGCCGTC A A A ACCTCGA-3 ’
[00134] SEQ ID NO: 59 depicts the nucleotide sequence of forward primer for Rv3603c.
[00135] 5 ’ -CCGGGACGCATGAGAGATG-3 ’
[00136] SEQ ID NO: 60 depicts the nucleotide sequence of reverse primer for Rv3603c.
[00137] 5 ’ - AGGCTC AAGGTGGGGATC AT- 3 ’
[00138] SEQ ID NO: 61 depicts the nucleotide sequence of forward primer for argS.
[00139] 5 ’ -GTGACCCCCGCTGACCTGGC-3 ’
[00140] SEQ ID NO: 62 depicts the nucleotide sequence of reverse primer for argS.
[00141] 5’-TCACATTCGCTCCGGTGCGG-3’
[00142] SEQ ID NO: 63 depicts the nucleotide sequence of forward primer for argS.
[00143] 5 ’ -CAAC ATGCGGCTGGAAACC-3 ’
[00144] SEQ ID NO: 64 depicts the nucleotide sequence of reverse primer for argS.
[00145] 5 ’ - AATTCC AGGTTGACCTTGCG-3 ’
[00146] SEQ ID NO: 65 depicts the nucleotide sequence of forward primer for kdpD.
[00147] 5 ’ -GTGACGTTGCTCTTCGCCGA-3 ’
[00148] SEQ ID NO: 66 depicts the nucleotide sequence of reverse primer for kdpD.
[00149] 5’-TCATGGGCGGTCCTCGGGAG-3 ’
[00150] SEQ ID NO: 67 depicts the nucleotide sequence of forward primer for kdpD.
[00151] 5 ’ - AAGTGCTGGATGTTGACCGT-3 ’
[00152] SEQ ID NO: 68 depicts the nucleotide sequence of reverse primer for kdpD.
[00153] 5’-AACACACCTGGCAGCAAGAA-3 ’
[00154] SEQ ID NO: 69 depicts the nucleotide sequence of forward primer for esxW.
[00155] 5 ’ -GGTC ATGTGTCCTCCTGAGT-3 ’
[00156] SEQ ID NO: 70 depicts the nucleotide sequence of reverse primer for esxW.
[00157] 5 ’ -TCGACGATTCA A AGGGAGG A-3 ’
[00158] SEQ ID NO: 71 depicts the nucleotide sequence of forward primer for esxW.
[00159] 5 ’ -GGCGTGCACCTC AAAACG-3 ’
[00160] SEQ ID NO: 72 depicts the nucleotide sequence of reverse primer for esxW.
[00161] 5 ’ -TTCGACGATTCAAAGGGAGGA-3 ’
[00162] SEQ ID NO: 73 depicts the nucleotide sequence of forward primer for Rvl520.
[00163] 5 ’ -GTGAGTATCGTCTCGATCTC-3 ’
[00164] SEQ ID NO: 74 depicts the nucleotide sequence of reverse primer for Rvl520.
[00165] 5 ’ -CT AGGCATGTGATCCGCGGA-3 ’
[00166] SEQ ID NO: 75 depicts the nucleotide sequence of forward primer for Rvl520.
[00167] 5’-CATGTTGCCCGAGACGATGG-3 ’
[00168] SEQ ID NO: 76 depicts the nucleotide sequence of reverse primer for Rvl520.
[00169] 5 ’ -GCCTCGTGTCTCCC AAAACTT-3 ’
[00170] SEQ ID NO: 77 depicts the nucleotide sequence of forward primer for IppB.
[00171] 5’-TTGATCGCACCACAACCGATTCCCCGA-3’
[00172] SEQ ID NO: 78 depicts the nucleotide sequence of reverse primer for IppB.
[00173] 5’-TCACGGGGTTGGCGTCGAGGTC -3’
[00174] SEQ ID NO: 79 depicts the nucleotide sequence of forward primer for IppB.
[00175] 5’-CGGCGACTGTTTTCTGTTGCAGAAGGTGCT-3’
[00176] SEQ ID NO: 80 depicts the nucleotide sequence of reverse primer for IppB.
[00177] 5 ’ -TC ACGGGGTTGGCGTCGAGGT-3 ’
[00178] Amplicons obtained from PCR using gene specific primer set (SEQ ID NO: 17 and SEQ ID NO: 18) and mutation specific primer set (SEQ ID NO: 19 and SEQ ID NO: 20) for moeY gene (SEQ ID NO: 1) comprises a nucleotide sequence having a sequence:
[00179] GCGAAGCGATGGGCCTATTATCCCTGGCGGCGCATGGTTGTTGC CATTCTGGGTCTCCGGGGGTTCCGTGCCGTGCGCTTGGACCGCAACAGG CACCTCATCACCG
[00180] Amplicons obtained from PCR using gene specific primer set (SEQ ID NO: 21 and SEQ ID NO: 22) and mutation specific primer set (SEQ ID NO: 23 and SEQ ID NO: 24) for Rv2957 gene (SEQ ID NO: 2) comprises a nucleotide sequence having a sequence:
[00181] ACGATCGTCGACAAGGAGTTTTTGAAGCGGCTGCCGATGTCCAC GAGACTCGGCATAAGGCTGGTCATAGTTCTGGTGCGCAGGTGAAGCGG CTGCCGATGTCCACGAGACTCGGCATAAGGCTGGTCATAGTTCTGGTGC GCAGG
[00182] Amplicons obtained from PCR using gene specific primer set (SEQ ID NO: 25 and SEQ ID NO: 26) and mutation specific primer set (SEQ ID NO: 27 and SEQ ID NO: 28) for Rv0177 gene (SEQ ID NO: 3) comprises a nucleotide sequence having a sequence:
[00183] CGGATCTCCCATGAATCGCACCAGCGAGCAGCGCACAAGGATA TCGTGATGCTCAGTGATGTCCGATCTTTCATGACCATGTTCACGTCACC GGATCC
[00184] Amplicons obtained from PCR using gene specific primer set (SEQ ID NO: 29 and SEQ ID NO: 30) and mutation specific primer set (SEQ ID NO: 31 and SEQ ID NO: 32) for Rv3727 gene (SEQ ID NO: 4) comprises a nucleotide sequence having a sequence:
[00185] TTCTGGAAAAACGTCCGCCTGCCGGAAGGAACCCGCTACTTCTG TCAATAACCTGGAGTGATGTGGAAACGCCCGGACCTGTTTTTGATCGGC [00186] Amplicons obtained from PCR using gene specific primer set (SEQ ID NO: 33 and SEQ ID NO: 34) and mutation specific primer set (SEQ ID NO: 35 and SEQ ID NO: 36) for Rv2522c gene (SEQ ID NO: 5) comprises a nucleotide sequence having a sequence:
[00187] GCTTTGACGGTGTCGCTACGCGGAATGGCCGACTGCGTCGTCGA GGTCGCCACCCTCGACCACGGGCTGCACTCCGGGTTGTGGGGCGGCGTC GTTCCCGACGCGCTGACCGTGCTAGTGCGGCTGTTGG
[00188] Amplicons obtained from PCR using gene specific primer set (SEQ ID NO: 37 and SEQ ID NO: 38) and mutation specific primer set (SEQ ID NO: 39 and SEQ ID NO: 40) for Rv0260c gene (SEQ ID NO: 6) comprises a nucleotide sequence having a sequence:
[00189] CGTTGATCCGAAAGGGCGTCCCGACGTCGGCTCCCGAGCGAAT GCGGTTGGGAGCCTTAGCCCGCCACATTGCCGAGGAGCTGCCGCTGCTG GGTTCGTGCACGTTCAAAG
[00190] Amplicons obtained from PCR using gene specific primer set (SEQ ID NO: 41 and SEQ ID NO: 42) and mutation specific primer set (SEQ ID NO: 43 and SEQ ID NO: 44) for Rv3770c gene (SEQ ID NO: 7) comprises a nucleotide sequence having a sequence:
[00191] GATGGTCCCCTCCTGACCATGGGCTGGCTGCTGGCGCGAGCGCT CACGGGCGAGCCCGCGGGCGCCCTCGGCCTGACCGTCCAGGTGCTCTG GGGACAGCTCA
[00192] Amplicons obtained from PCR using gene specific primer set (SEQ ID NO: 45 and SEQ ID NO: 46) and mutation specific primer set (SEQ ID NO: 47 and SEQ ID NO: 48) for Rv2743c gene (SEQ ID NO: 8) comprises a nucleotide sequence having a sequence:
[00193] TCCGAACGCGGGTTCTTCTCGCTGTTGGGTGTCATGGAGCGGGG CGCCATGTTGCCGGCGGACGAGATCCGCGACCTAACG
[00194] Amplicons obtained from PCR using gene specific primer set (SEQ ID NO: 49 and SEQ ID NO: 50) and mutation specific primer set (SEQ ID NO: 51 and SEQ ID NO: 52) for Rvl358 gene (SEQ ID NO: 9) comprises a nucleotide sequence having a sequence:
[00195] CGAACGGAGCATTGGCACTATCTCGCGACGTTACCGCCGAGGC CGAGGTGGCAAACGATGTCGTTACTAAGGTACTCGGTTTGTATGTCGAA GCCATGGCA
[00196] Amplicons obtained from PCR using gene specific primer set (SEQ ID NO: 53 and SEQ ID NO: 54) and mutation specific primer set (SEQ ID NO: 55 and SEQ ID NO: 56) for Rv0845 gene (SEQ ID NO: 10) comprises a nucleotide sequence having a sequence:
[00197] GCCTGTACGCTAGTCGGATTCGCAGTCGCGGTGCTGGGAGACCC CGTGATGCTGCGCGCGATTGGATGGCCCGAGACAATATTTCGGTTCGCG CTCTATG
[00198] Amplicons obtained from PCR using gene specific primer set (SEQ ID NO: 57 and SEQ ID NO: 58) and mutation specific primer set (SEQ ID NO: 59 and SEQ ID NO: 60) for Rv3603c gene (SEQ ID NO: 11) comprises a nucleotide sequence having a sequence:
[00199] AGGCTCAAGGTGGGGATCATCTCGGCTGGCCGGGTCGGCACCG CGCTAGGGGTCGCGCTGCAGCGCGCCGACCATGTTGTGGTGGCGTGCA GCGCCATCTCTCATGCGTCCCGG
[00200] Amplicons obtained from PCR using gene specific primer set (SEQ ID NO: 61 and SEQ ID NO: 62) and mutation specific primer set (SEQ ID NO: 63 and SEQ ID NO: 64) for argS gene (SEQ ID NO: 12) comprises a nucleotide sequence having a sequence:
[00201] CAACATGCGGCTGGAAACCGCCGCCCAGGCTAAAGTCGTTACC AGCGTTATCGACGCCGGCCACAGCTACGGTCACTCGCTGCTGCTGGCCG GGCGCAAGGTCAACCTGGAATT
[00202] Amplicons obtained from PCR using gene specific primer set (SEQ ID NO: 65 and SEQ ID NO: 66) and mutation specific primer set (SEQ ID NO: 67 and SEQ ID NO: 68) for kdpD gene (SEQ ID NO: 13) comprises a nucleotide sequence having a sequence:
[00203] AACACACCTGGCAGCAAGAACCCCAAGCGCTGGCAGGACGTTC AGGAAATCCTCGACGCCGGCATCACGGTGATCTCGACGGTCAACATCC AGCACTT
[00204] Amplicons obtained from PCR using gene specific primer set (SEQ ID NO: 69 and SEQ ID NO: 70) and mutation specific primer set (SEQ ID NO: 71 and SEQ ID NO: 72) for esxW gene (SEQ ID NO: 14) comprises a nucleotide sequence having a sequence:
[00205] TTCGACGATTCAAAGGGAGGAATTCATATGACCTCGCGTTTTAT GACGGATCCGCACGCGATGCGGGACATGGCGGGCCGTTTTGAGGTGCA CGCC
[00206] Amplicons obtained from PCR using gene specific primer set (SEQ ID NO: 73 and SEQ ID NO: 74) and mutation specific primer set (SEQ ID NO: 75 and SEQ ID NO: 76) for Rvl520 gene (SEQ ID NO: 15) comprises a nucleotide sequence having a sequence:
[00207] CATGTTGCCCGAGACGATGGCGGTCTACCGTCGCCACGCTCACG GTATTTGGCATTCCGCGTACACTGACCGCCGAAAGTTTTGGGAGACACG AGGC
[00208] Amplicons obtained from PCR using gene specific primer set (SEQ ID NO: 77 and SEQ ID NO: 78) and mutation specific primer set (SEQ ID NO: 79 and SEQ ID NO: 80) for IppB gene (SEQ ID NO: 16) comprises a nucleotide sequence having a sequence:
[00209] CGGCGACTGTTTTCTGTTGCAGAAGGTGCTCGACCTGCCGGCCG GACAACTCCCCCCCGAACCACCCATTTGGCCGACGACCTCGACGCCAAC CCCGTGA
Definitions
[00210] For convenience, before further description of the present disclosure, certain terms employed in the specification, and examples are delineated here. These definitions should be read in the light of the remainder of the disclosure and understood as by a person of skill in the art. The terms used herein have the meanings recognized and known to those of skill in the art, however, for convenience and completeness, particular terms and their meanings are set forth below.
[00211] The articles “a”, “an” and “the” are used to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article.
[00212] Throughout this specification, unless the context requires otherwise the word “comprise”, and variations such as “comprises” and “comprising” are used in the inclusive, open sense, and will be understood to imply the inclusion of a stated element or step or group of element or steps but not the exclusion of any other element or step or group of element or steps. It is not intended to be construed as “consists of only”.
[00213] The term “including” is used to mean “including but not limited to”. “Including” and “including but not limited to” are used interchangeably.
[00214] The term “tuberculosis” refers to a bacterial infection that affects primarily the lungs. The bacterium that causes tuberculosis is Mycobacterium tuberculosis.
[00215] The term “polynucleotide” refers to a combination of nucleotide monomers, which are connected to each other through covalent bonds as in DNA or RNA.
[00216] For the purposes of the present disclosure, the term “a panel of genetic variations” or “genetic variations panel” is used to define a group of genetic variations, mainly mutations that reflect the drug resistance status of M. tuberculosis. [00217] The term “mutation” refers to an alteration in the DNA sequence that makes up a gene. Mutations could be either a substitution, insertion, or deletion. In the present disclosure the term “nucleotide substitution” refers to the exchange of one base for another (i.e., a change in a single “chemical letter” such as switching an A to a G). For example, the representation “T30C” refers to the substitution of the nucleotide Thymine, at a position 30 on the nucleotide fragment, with the nucleotide Cytosine. In another example, the representation “G1177C” refers to the substitution of the nucleotide Guanine, at a position 1117 on the nucleotide fragment, with the nucleotide Cytosine. Similar representations for nucleotide substitutions have been referred to herein, the meaning of which would be apparent to a person skilled in the art in light of the present disclosure.
[00218] The term “antibiotic” refers to a substance used in the treatment of bacterial infections for eg: M. tuberculosis infections. It refers to a substance that is capable of inhibiting or killing bacteria. In the present disclosure, the term “antibiotic” refers to those substances capable of killing M. tuberculosis.
[00219] The term “resistance” refers to the ability of a bacteria to evade the effect of antibiotics. In the present disclosure the absence of resistance in M. tuberculosis is referred to as “susceptible”.
[00220] The term “amplicon” refers to the nucleotide product of amplification reactions, i.e., PCR product. In the context of the present disclosure the terms “amplicon” and “PCR product” are used interchangeably.
[00221] The term “subject” refers to any mammal. It refers to a mammal whose blood or tissue sample has been taken for analysis. It particularly refers to mammals who are having or suspected of having bacterial infections. In an example, the subject
is a mammal having M. tuberculosis infection. The exemplification is based on humans used as subjects.
[00222] Ratios, concentrations, amounts, and other numerical data may be presented herein in a range format. It is to be understood that such range format is used merely for convenience and brevity and should be interpreted flexibly to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited.
[00223] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the disclosure, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference.
[00224] The present disclosure is not to be limited in scope by the specific embodiments described herein, which are intended for the purposes of exemplification only. Functionally equivalent products, compositions, and methods are clearly within the scope of the disclosure, as described herein.
[00225] As discussed in the background section, there are various limitations associated with the conventional materials, and methods for detecting drug resistant tuberculosis. The traditional drug susceptibility testing for tuberculosis has been conducted phenotypically using culture-based methods that takes several weeks due to the slow growing nature of M. tuberculosis. More recently, molecular diagnostics that detect mutations associated with resistance to TB drugs such as the Xpert MTB/RIF cartridge-based system (Cepheid), and Hain line probe assay (LPA) (Hain Lifescience) have been employed for determining drug resistant tuberculosis. Though these molecular methods offer improved speed and ease of detection, they are restricted to detecting resistance towards isoniazid and rifampicin, the two major first line drugs. Moreover, these tests are limited to widely occurring resistance mutations and do not account for rare resistance causing mutations even in the case of isoniazid and rifampicin. lienee, identification of a greater number of novel
mutations are required, which are associated with drug resistance in M. tuberculosis against the wide spectrum of antibiotics used as a part of T B treatment regime.
[00226] To address the aforementioned problems, the present disclosure discloses polynucleotide fragments of M. tuberculosis, wherein presence of specific nucleotide substitution results in resistance to antibiotics such as pyrazinamide, rifampicin, ethambutol, and streptomycin. The present disclosure discloses a method for determining whether a subject infected with M. tuberculosis is resistant or susceptible to a specific antibiotic for treatment of tuberculosis. A method is also described for preparing a treatment regimen for a subject infected with M. tuberculosis based on the identification of the mutation. The present disclosure also provides a kit and a panel of polynucleotides for determining whether a subject infected with M. tuberculosis is resistant or susceptible to a specific antibiotic for treatment of tuberculosis.
[00227] As an outcome of this, the present disclosure solves the problems existing in the art by providing novel nucleotide substitutions in gene targets against antibiotics that are helpful in determining drug resistant tuberculosis and as well as in guiding treatment regimens.
[00228] In an embodiment of the present disclosure, there is provided a polynucleotide fragment of M. tuberculosis, having a nucleic acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16, wherein: SEQ ID NO: 1 comprises a nucleotide substitution at C216T; SEQ ID NO: 2 comprises a nucleotide substitution at G705A; SEQ ID NO: 3 comprises a nucleotide substitution at A174C; SEQ ID NO: 4 comprises a nucleotide substitution at G1177C; SEQ ID NO: 5 comprises a nucleotide substitution at C674G; SEQ ID NO: 6 comprises a nucleotide substitution at C771T; SEQ ID NO: 7 comprises a nucleotide substitution at C343T; SEQ ID NO: 8 comprises a nucleotide substitution at G466T; SEQ ID NO: 9 comprises a nucleotide substitution at CCGA[2522]C; SEQ ID NO: 10 comprises a nucleotide substitution at T533G; SEQ ID NO: 11 comprises a nucleotide substitution at
Cl 12T; SEQ ID NO: 12 comprises a nucleotide substitution at G355A; SEQ ID NO: 13 comprises a nucleotide substitution at C383A; SEQ ID NO: 14 comprises a nucleotide substitution at T30C; SEQ ID NO: 15 comprises a nucleotide substitution at C643T; and SEQ ID NO: 16 comprises a nucleotide substitution at T633T; wherein the presence of nucleotide substitution indicates the resistance to antibiotic for treatment of tuberculosis.
[00229] Accordingly, embodiments herein provide a panel of nucleotide fragments for determining antibiotic resistance in a subject. In an embodiment, the panel comprises at least one polynucleotide fragment of M. tuberculosis, having a nucleic acid sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16, wherein: SEQ ID NO: 1 comprises a nucleotide substitution at C216T; SEQ ID NO: 2 has a nucleotide substitution at G705A; SEQ ID NO: 3 comprises a nucleotide substitution at A174C; SEQ ID NO: 4 comprises a nucleotide substitution at G1177C; SEQ ID NO: 5 comprises a nucleotide substitution at C674G; SEQ ID NO: 6 comprises a nucleotide substitution at C771T; SEQ ID NO: 7 comprises a nucleotide substitution at C343T; SEQ ID NO: 8 comprises a nucleotide substitution at G466T; SEQ ID NO: 9 comprises a nucleotide substitution at CCGA[2522]C; SEQ ID NO: 10 comprises a nucleotide substitution at T533G; SEQ ID NO: 11 comprises a nucleotide substitution atC112T; SEQ ID NO: 12 comprises a nucleotide substitution at G355A; SEQ ID NO: 13 comprises a nucleotide substitution at C383A; SEQ ID NO: 14 comprises a nucleotide substitution at T30C; SEQ ID NO: 15 comprises a nucleotide substitution at C643T; and SEQ ID NO: 16 comprises a nucleotide substitution at T633T; wherein, the presence of nucleotide substitution indicates resistance to antibiotic for treatment of tuberculosis.
[00230] In an embodiment of the present disclosure, there is provided a polynucleotide fragment of M. tuberculosis as described herein, wherein the nucleotide substitution in the sequence as set forth in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3 indicates resistance to the antibiotic pyrazinamide.
[00231] In an embodiment, there is provided a panel for determining resistance to the antibiotic pyrazinamide, said panel comprising a polynucleotide fragment of M. tuberculosis comprising a nucleotide substitution in the sequence as set forth in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, wherein: SEQ ID NO: 1 comprises a nucleotide substitution at C216T; SEQ ID NO: 2 comprises a nucleotide substitution at G705A; and SEQ ID NO: 3 comprises a nucleotide substitution at A174C.
[00232] In an embodiment of the present disclosure, there is provided a polynucleotide fragment of M. tuberculosis as described herein, wherein the nucleotide substitution in the sequence as set forth in SEQ ID NO: 4 indicates resistance to the antibiotic rifampicin.
[00233] In an embodiment, there is provided a panel for determining resistance to the antibiotic rifampicin, said panel comprising a polynucleotide fragment of M. tuberculosis comprising a nucleotide substitution in the sequence as set forth in SEQ ID NO: 4, wherein SEQ ID NO: 4 comprises a nucleotide substitution at G1177C.
[00234] In an embodiment of the present disclosure, there is provided a polynucleotide fragment of M. tuberculosis as described herein, wherein the nucleotide substitution in the sequence as set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NOG, SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 13 indicates resistance to the antibiotic ethambutol.
[00235] In an embodiment, there is provided a panel for determining resistance to the antibiotic ethambutol, said panel comprising a polynucleotide fragment of M. tuberculosis comprising a nucleotide substitution in the sequence as set forth in SEQ ID NO:1, SEQ ID NOG, SEQ ID NOG, SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 13, wherein SEQ ID NO: 1 comprises a nucleotide substitution at C216T; SEQ ID NO: 2 comprises a nucleotide substitution at G705A; SEQ ID NO: 3 comprises a nucleotide substitution at A174C; SEQ ID NO: 5 comprises a nucleotide substitution at C674G; SEQ ID NO: 6 comprises a nucleotide substitution at C771T; SEQ ID NO: 7 comprises a nucleotide substitution at C343T; SEQ ID NO: 8 comprises a nucleotide substitution at G466T; SEQ ID NO: 9
comprises a nucleotide substitution at CCGA[2522]C; SEQ ID NO: 10 comprises a nucleotide substitution at T533G; SEQ ID NO: 11 comprises a nucleotide substitution at C112T; SEQ ID NO: 12 comprises a nucleotide substitution at G355A; and SEQ ID NO: 13 comprises a nucleotide substitution at C383A.
[00236] In an embodiment of the present disclosure, there is provided a polynucleotide fragment of M. tuberculosis as described herein, wherein nucleotide substitution in the sequence as set forth in SEQ ID NO: 4, SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16 indicates resistance to the antibiotic streptomycin.
[00237] In an embodiment, there is provided a panel for determining resistance to the antibiotic streptomycin, said panel comprising a polynucleotide fragment of M. tuberculosis comprising a nucleotide substitution in the sequence as set forth in SEQ ID NO: 4, SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16, wherein SEQ ID NO: 4 comprises a nucleotide substitution at G1177C; SEQ ID NO: 14 comprises a nucleotide substitution at T30C; SEQ ID NO: 15 comprises a nucleotide substitution at C643T; and SEQ ID NO: 16 comprises a nucleotide substitution at T633C.
[00238] In an embodiment of the present disclosure, there is provided a method for determining resistance or susceptibility of a subject to a specific antibiotic for the treatment of tuberculosis, the method comprising the steps of: (a) obtaining a nucleic acid from a sample of the subject; and (b) sequencing the nucleic acid to detect the presence or absence of the substitutions as described herein; wherein, the presence of the substitution in the nucleic acid determines that the subject is resistant to the specific antibiotic.
[00239] In an embodiment of the present disclosure, there is provided a method for determining resistance or susceptibility of a subject to a specific antibiotic for treatment of tuberculosis, the said method comprising the steps of: (a) obtaining a nucleic acid from a sample of the subject; and (b) performing a PCR amplification with primer sets to obtain an amplicon and sequencing the amplicon to detect for the presence or absence of the substitutions as described herein, wherein the primer set comprises primers having a sequence selected from sequence as set forth in: SEQ ID NO: 17 and SEQ ID NO: 18 for detecting mutation in SEQ ID NO: 1; SEQ ID NO: 21 and SEQ ID NO: 22 for detecting mutation in SEQ ID NO: 2; SEQ ID NO: 25
and SEQ ID NO: 26 for detecting mutation in SEQ ID NO: 3; SEQ ID NO: 29 and SEQ ID NO: 30 for detecting mutation in SEQ ID NO: 4; SEQ ID NO: 33 and SEQ ID NO: 34 for detecting mutation in SEQ ID NO: 5; SEQ ID NO: 37 and SEQ ID NO: 38 for detecting mutation in SEQ ID NO: 6; SEQ ID NO: 41 and SEQ ID NO: 42 for detecting mutation in SEQ ID NO: 7; SEQ ID NO: 45 and SEQ ID NO: 46 for detecting mutation in SEQ ID NO: 8; SEQ ID NO: 49 and SEQ ID NO: 50 for detecting mutation in SEQ ID NO: 9; SEQ ID NO: 53 and SEQ ID NO: 54 for detecting mutation in SEQ ID NO: 10; SEQ ID NO: 57 and SEQ ID NO: 58 for detecting mutation in SEQ ID NO: 11; SEQ ID NO: 61 and SEQ ID NO: 62 for detecting mutation in SEQ ID NO: 12; SEQ ID NO: 65 and SEQ ID NO: 66 for detecting mutation in SEQ ID NO: 13; SEQ ID NO: 69 and SEQ ID NO: 70 for detecting mutation in SEQ ID NO: 14; SEQ ID NO: 73 and SEQ ID NO: 74 for detecting mutation in SEQ ID NO: 15; and SEQ ID NO: 77 and SEQ ID NO: 78 for detecting mutation in SEQ ID NO: 16; or combinations thereof; wherein, the presence of the substitution in the amplicon indicates the resistance to a specific antibiotic.
[00240] In an embodiment, there is provided a method for determining resistance or susceptibility of a subject to a specific antibiotic for treatment of tuberculosis, wherein the primer set comprises primers having a sequence selected from sequence as set forth in: SEQ ID NO: 19 and SEQ ID NO: 20 for detecting mutation in SEQ ID NO: 1; SEQ ID NO: 23 and SEQ ID NO: 24 for detecting mutation in SEQ ID NO: 2; SEQ ID NO: 27 and SEQ ID NO: 28 for detecting mutation in SEQ ID NO: 3; SEQ ID NO: 31 and SEQ ID NO: 32 for detecting mutation in SEQ ID NO: 4; SEQ ID NO: 35 and SEQ ID NO: 36 for detecting mutation in SEQ ID NO: 5; SEQ ID NO: 39 and SEQ ID NO: 40 for detecting mutation in SEQ ID NO: 6; SEQ ID NO: 43 and SEQ ID NO: 44 for detecting mutation in SEQ ID NO: 7; SEQ ID NO: 47 and SEQ ID NO: 48 for detecting mutation in SEQ ID NO: 8; SEQ ID NO: 51 and SEQ ID NO: 52 for detecting mutation in SEQ ID NO: 9; SEQ ID NO: 55 and SEQ ID NO: 56 for detecting mutation in SEQ ID NO: 10; SEQ ID NO: 59 and SEQ ID NO: 60 for detecting mutation in SEQ ID NO: 11; SEQ ID NO: 63 and SEQ ID NO: 64 for detecting mutation in SEQ ID NO: 12; SEQ ID NO: 67 and SEQ ID NO:
68 for detecting mutation in SEQ ID NO: 13; SEQ ID NO: 71 and SEQ ID NO: 72 for detecting mutation in SEQ ID NO: 14; SEQ ID NO: 75 and SEQ ID NO: 76 for detecting mutation in SEQ ID NO: 15; SEQ ID NO: 79 and SEQ ID NO: 80 for detecting mutation in SEQ ID NO: 16; or combinations thereof; wherein the presence of the substitution in the amplicon indicates the resistance to a specific antibiotic.
[00241] In an embodiment of the present disclosure there is provided a method for determining resistance or susceptibility of a subject to a specific antibiotic for treatment of tuberculosis as described herein, wherein the sample is selected from the group consisting of blood, sputum, and spinal fluids.
[00242] In an embodiment of the present disclosure there is provided a method for determining resistance or susceptibility of a subject to a specific antibiotic for treatment of tuberculosis as described herein, wherein the antibiotic is selected from pyrazinamide, rifampicin, ethambutol, streptomycin, or combinations thereof.
[00243] In an embodiment of the present disclosure, there is provided a method of preparing a treatment regimen for a subject infected with M. tuberculosis, said method comprising the steps of: (a) determining whether the subject infected with tuberculosis is resistant to an antibiotic using the method as described herein; and (b) selecting an antibiotic for which the subject having tuberculosis is not resistant as determined in step (a) for preparing a treatment regimen, wherein the antibiotic is selected from the group consisting of isoniazid, rifampicin/rifampin, ethambutol, pyrazinamide, rifabutin, rifapentine, streptomycin, moxifloxacin, ciprofloxacin, levofloxacin, ofloxacin, p-aminosalicylic acid, ethionamide, prothionamide, cycloserine, terizidone, capreomycin, kanamycin, amikacin, linezolid, delamanid, pretomanid, clofazimine and combinations thereof.
[00244] In an embodiment of the present disclosure, there is provided a method for determining whether a subject infected with M. tuberculosis is resistant or susceptible to a specific antibiotic for treatment of tuberculosis, wherein the subject is human.
[00245] In an embodiment of the present disclosure, there is provided a method of preparing a treatment regimen for a subject infected with M. tuberculosis, wherein the subject is human.
[00246] In an embodiment of the present disclosure, there is provided a kit for determining resistance or susceptibility of a subject to a specific antibiotic for the treatment of tuberculosis, comprising one or more of primer set, wherein the primer set comprises primers having a sequence selected from sequence as set forth in: SEQ ID NO: 17 and SEQ ID NO: 18 for detecting mutation in SEQ ID NO: 1; SEQ ID NO: 21 and SEQ ID NO: 22 for detecting mutation in SEQ ID NO: 2; SEQ ID NO: 25 and SEQ ID NO: 26 for detecting mutation in SEQ ID NO: 3; SEQ ID NO: 29 and SEQ ID NO: 30 for detecting mutation in SEQ ID NO: 4; SEQ ID NO: 33 and SEQ ID NO: 34 for detecting mutation in SEQ ID NO: 5; SEQ ID NO: 37 and SEQ ID NO: 38 for detecting mutation in SEQ ID NO: 6; SEQ ID NO: 41 and SEQ ID NO: 42 for detecting mutation in SEQ ID NO: 7; SEQ ID NO: 45 and SEQ ID NO:
46 for detecting mutation in SEQ ID NO: 8; SEQ ID NO: 49 and SEQ ID NO: 50 for detecting mutation in SEQ ID NO: 9; SEQ ID NO: 53 and SEQ ID NO: 54 for detecting mutation in SEQ ID NO: 1; SEQ ID NO: 57 and SEQ ID NO: 58 for detecting mutation in SEQ ID NO: 11; SEQ ID NO: 61 and SEQ ID NO: 62 for detecting mutation in SEQ ID NO: 12; SEQ ID NO: 65 and SEQ ID NO: 66 for detecting mutation in SEQ ID NO: 13; SEQ ID NO: 69 and SEQ ID NO: 70 for detecting mutation in SEQ ID NO: 14; SEQ ID NO: 73 and SEQ ID NO: 74 for detecting mutation in SEQ ID NO: 15; and SEQ ID NO: 77 or SEQ ID NO: 78 for detecting mutation in SEQ ID NO: 16.
[00247] In another embodiment of the present disclosure, there is provided a kit, wherein the primer set comprises primers having a sequence selected from sequence as set forth in: SEQ ID NO: 19 and SEQ ID NO: 20 for detecting mutation in SEQ ID NO: 1; SEQ ID NO: 23 and SEQ ID NO: 24 for detecting mutation in SEQ ID NO: 2; SEQ ID NO: 27 and SEQ ID NO: 28 for detecting mutation in SEQ ID NO: 3; SEQ ID NO: 31 and SEQ ID NO: 32 for detecting mutation in SEQ ID NO: 4; SEQ ID NO: 35 and SEQ ID NO: 36 for detecting mutation in SEQ ID NO: 5; SEQ ID NO: 39 and SEQ ID NO: 40 for detecting mutation in SEQ ID NO: 6; SEQ ID NO: 43 and SEQ ID NO: 44 for detecting mutation in SEQ ID NO: 7; SEQ ID NO:
47 and SEQ ID NO: 48 for detecting mutation in SEQ ID NO: 8; SEQ ID NO: 51 and SEQ ID NO: 52 for detecting mutation in SEQ ID NO: 9; SEQ ID NO: 55 and
SEQ ID NO: 56 for detecting mutation in SEQ ID NO: 10; SEQ ID NO: 59 and SEQ ID NO: 60 for detecting mutation in SEQ ID NO: 11; SEQ ID NO: 63 and SEQ ID NO: 64 for detecting mutation in SEQ ID NO: 12; SEQ ID NO: 67 and SEQ ID NO: 68 for detecting mutation in SEQ ID NO: 13; SEQ ID NO: 71 and SEQ ID NO: 72 for detecting mutation in SEQ ID NO: 14; SEQ ID NO: 75 and SEQ ID NO: 76 for detecting mutation in SEQ ID NO: 15; or SEQ ID NO: 79 or SEQ ID NO: 80 for detecting mutation in SEQ ID NO: 16.
[00248] In some embodiments, the kit further comprises a DNA polymerase, extension nucleotides, and buffer.
[00249] In an embodiment, there is provided a method for determining whether a subject infected with M. tuberculosis is resistant or susceptible to a specific antibiotic for treatment of tuberculosis, the method comprising the steps of: a. obtaining a nucleic acid from a sample of the subject; and b. performing a PCR amplification with the kit as disclosed herein to obtain an amplicon and sequencing the amplicon to detect for the presence or absence of the substitutions.
[00250] In some embodiments, the kit may comprise other amplification reagents that are useful, necessary, or sufficient for practicing any of the methods described herein, as well as instructions, analysis software (e.g., that facilitates data collection, analysis, display, and reporting), computing devices, instruments, or other systems or components.
[00251] Although the subject matter has been described in considerable detail with reference to certain examples and implementations thereof, other implementations are possible.
EXAMPLES
[00252] The disclosure will now be illustrated with working examples, which is intended to illustrate the working of disclosure and not intended to take restrictively to imply any limitations on the scope of the present disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described
herein can be used in the practice of the disclosed methods and compositions, the exemplary methods, devices and materials are described herein. It is to be understood that this disclosure is not limited to particular methods, and experimental conditions described, as such methods and conditions may apply.
Example 1:
Identification of novel mutations for determining drug resistance in M. tuberculosis
[00253] The mutations as described here have been identified with the help of the system and the method as described in Indian Application no. 202041055641, which is included here in its entirety. A brief summary of the method is provided hereinbelow (Figure 1).
[00254] The method consists of three components- bioinformatics analysis, machine learning (ML) analysis, and artificial intelligence (Al) prediction tool. When a sample from a database/ hospital/ diagnostic lab is received, it goes through the bioinformatics and ML analysis first and then a patient antibiotic sensitivity report is generated and provided for that patient to the source of the sample. The data is however stored and used in the Al pipeline to analyse the whole genome sequence for novel targets and hotspots within the genome, that also show statistical significance in samples that are associated with resistance to drugs. The bioinformatics and ML analysis pipelines are briefly described below, followed by the description of the Al and the in silico and in vitro validations that can be done for such novel targets found.
Bioinformatic analysis:
[00255] An automated, sequencer agnostic bioinformatics pipeline was built for pathogen identification and variant calling. The pipeline can process raw Sequencing files in FASTQ format as well as Fast5 format to extract taxonomic information of the microorganisms present in the samples and further sorted to select reads belonging to Mycobacterium tuberculosis for variant calling. Low quality variants were marked separately as part of the analysis. The remaining variants detected in the aligned reads were parsed by the AarogyaAI® machine learning algorithm based
antibiotic resistance prediction as described earlier in the Indian patent application: 202041055641.
Machine Learning Model:
[00256] The analysis was done by the proprietary AarogyaAI® (Indian patent application: 202041055641) that combines bioinformatics analysis with a machine learning model, trained on 7000 whole genome sequenced (WGS) M. tuberculosis data. The data frame is trained based on logistic regression, random forest, extra trees, and gradient boosting trees classifier models. The best model is selected based on performance using Pycaret. The WGS samples used for training had a known drug susceptibility status, which was used as ground truth.
Artificial Intelligence (Al) Predictions:
[00257] The ML model chosen after training the system on the 7000 whole genome sequences of M. tb, is scrutinized for biases created by not just the known, but the unknown regions of the genome as well. Mutations of low frequency, collinear mutations (duplicate appearance), mutations with low importance and mutations with unique value were removed to have the essential mutations. In particular, the regions showing mutations maximally in all known resistant samples, and minimally in all known sensitive samples are considered as potential novel candidates for further investigation (Figure 2). A list of 50 such targets for five drugs, namely rifampicin, isoniazid, ethambutol, streptomycin and pyrazinamide was created. As a positive control, it was checked if the well-known mutations for these drugs are among the top candidates for resistance. After observing the presence of well-known mutations in the top 50 candidates, in silico validation was performed as described below.
In silico validation:
[00258] For the in-silico validation of each mutation, a 2X2 matrix was created.
[00259] This led to the calculation of percentage (%) of a mutation’s presence in resistant samples and percentage (%) of a mutation’s presence in sensitive samples. The following criteria was used as a threshold for these percentages:
[00260] % Of presence in resistant samples: >75%
[00261] % Of presence in sensitive samples: <15%
[00262] Only the mutations that met these criteria were taken forward (Figure 2). The well-known mutations identified using the described method are listed in Table
1.
[00263] Table 1
[00264] Thus, the novel mutations that were not listed in any of the reported WHO catalogues or any other research papers, obtained at the end of in silico validation analysis for the different antibiotics are listed in Table 2.
[00265] Table 2
Example 2:
In vitro validation
[00266] To study the effects of these individual mutations on resistance to the respective drugs, these mutations were introduced in a susceptible background (Mycobacterium tuberculosis strain H37rv) using genome editing that introduces point mutations. Upon successful mutant generation, sequencing was performed to ensure that no non-specific mutations were introduced during the process. The effect of these mutations was then tested on changes in MIC values for the respective drugs. Based on the change in MICs, the mutations were categorized as high level, midlevel, or low-level mutations. Further support was generated by complementing the mutations and seeing reversal in MIC values. The substitutions/mutations as described in the present disclosure are novel targets that can be used to determine if a subject infected with M. tuberculosis is resistant to a specific antibiotic which in turn will aid in providing the appropriate treatment to the subject in a timely manner.
Example 3:
Detection of antibiotic resistance in a subject infected with M. tuberculosis’.
[00267] The method for detecting antibiotic resistance in a TB patient is provided below:
[00268] Method 1: By direct sequencing of the nucleic acid obtained from patient sample, the steps of the method are: obtaining a sample, such as blood, sputum or spinal fluid from a subject who has been infected with M. tuberculosis; extracting nucleic acid from the sample using methods well known in the art; subjecting the nucleic acid to sequencing to check for the presence or absence of the substitutions as described hereinabove; wherein, the presence of the substitution in the nucleic acid determines that the subject is resistant to a specific antibiotic.
[00269] Method 2: By performing PCR (using Fusion High-Fidelity PCR Kit. Catalog Number; F553L) with gene specific or mutation specific primers: obtaining a sample, such as blood, sputum or spinal fluid from a subject who has been infected with M. tuberculosis; extracting nucleic acid from the sample using methods well known in the art; performing a PCR amplification (with conditions as mentioned in Table 3 and 4 for 25 cycles) with primer sets (gene specific or mutation specific as provided in Tables 3 and 4 below) to obtain an amplicon (as provided below in Tables 3 and 4) and sequencing the amplicon to detect for the presence or absence of the substitutions as described hereinabove, wherein, the presence of the substitution in the nucleic acid determines that the subject is resistant to a specific antibiotic.
[00270] The details of the primers are provided in the Table below:
Table 3: Gene specific primers:
Table 4: Mutation specific primers:
Example 4:
[00271] Once the mutation is identified in the patient sample, the information can be used for designing a treatment regimen for the patient. The method is described below:
Step 1: determining whether a subject infected with M. tuberculosis is resistant to an antibiotic using any one of the methods described in Example 3;
Step 2: selecting an antibiotic for which the subject having tuberculosis is not resistant as determined in step(l) for preparing a treatment regimen, wherein the antibiotic is selected from the group consisting of isoniazid, rifampicin/rifampin, ethambutol, pyrazinamide, rifabutin, rifapentine, streptomycin, moxifloxacin, ciprofloxacin, levofloxacin, ofloxacin, p- aminosalicylic acid, ethionamide, prothionamide, cycloserine, terizidone, capreomycin, kanamycin, amikacin, linezolid, delamanid, pretomanid, clofazimine and combinations thereof.
Advantages of the present disclosure
[00272] The present disclosure provides polypeptide fragments having novel mutations for determining whether a subject who is infected with M. tuberculosis is resistant or susceptible to a specific antibiotic.
[00273] The present disclosure has the following advantages:
(a) The method for determining whether a subject infected with M. tuberculosis is resistant or susceptible to a specific antibiotic for treatment of tuberculosis as described herein is time saving and accurate when compared to the conventional drug susceptibility testing. (b) The novel mutations as described here will offer guidance in selection of appropriate TB treatment regimen in a timely manner and will add to the already existing catalogues for drug resistance tuberculosis.
(c) Every mutation disclosed herein is detected both amongst resistant as well as sensitive samples suggesting the importance of incorporating these in the databases to improve genomic drug susceptibility reports.
Claims
1. A polynucleotide fragment of M. tuberculosis having a nucleic acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16, wherein:
SEQ ID NO: 1 comprises a nucleotide substitution at C216T;
SEQ ID NO: 2 comprises a nucleotide substitution at G705A; SEQ ID NO: 3 comprises a nucleotide substitution at A174C; SEQ ID NO: 4 comprises a nucleotide substitution at G1177C; SEQ ID NO: 5 comprises a nucleotide substitution at C674G;
SEQ ID NO: 6 comprises a nucleotide substitution at C771T; SEQ ID NO: 7 comprises a nucleotide substitution at C343T; SEQ ID NO: 8 comprises a nucleotide substitution at G466T;
SEQ ID NO: 9 comprises a nucleotide substitution at CCGA[2522]C;
SEQ ID NO: 10 comprises a nucleotide substitution at T533G;
SEQ ID NO: 11 comprises a nucleotide substitution at Cl 12T; SEQ ID NO: 12 comprises a nucleotide substitution at G355A; SEQ ID NO: 13 comprises a nucleotide substitution at C383A; SEQ ID NO: 14 comprises a nucleotide substitution at T30C;
SEQ ID NO: 15 comprises a nucleotide substitution at C643T; and
SEQ ID NO: 16 comprises a nucleotide substitution at T633C; wherein the presence of nucleotide substitution indicates resistance to antibiotic for treatment of tuberculosis.
2. The polynucleotide fragment as claimed in claim 1, wherein the nucleotide substitution in the sequence as set forth in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3 indicates resistance to the antibiotic pyrazinamide.
3. The polynucleotide fragment as claimed in claim 1, wherein the nucleotide substitution in the sequence as set forth in SEQ ID NO: 4 indicates resistance to the antibiotic rifampicin.
The polynucleotide fragment as claimed in claim 1, wherein the nucleotide substitution in the sequence as set forth in SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 13 indicates resistance to the antibiotic ethambutol. The polynucleotide fragment as claimed in claim 1, wherein nucleotide substitution in the sequence as set forth in SEQ ID NO: 4, SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16 indicates resistance to the antibiotic streptomycin. A method for determining resistance or susceptibility of a subject to a specific antibiotic for the treatment of tuberculosis, the method comprising the steps of: a. obtaining a nucleic acid from a sample of the subject; and b. sequencing the nucleic acid to detect the presence or absence of the substitutions as claimed in claims 1 to 5; wherein the presence of the substitution in the nucleic acid determines that the subject is resistant to the specific antibiotic. A method for determining resistance or susceptibility of a subject to a specific antibiotic for treatment of tuberculosis, the method comprising the steps of: a. obtaining a nucleic acid from a sample of the subject; and b. performing a PCR amplification with primer set to obtain an amplicon and sequencing the amplicon to detect for the presence or absence of the substitutions as claimed in claims 1 to 5, wherein the primer set comprises primers having a sequence selected from sequence as set forth in:
- SEQ ID NO: 17 and SEQ ID NO: 18 for detecting mutation in SEQ ID NO: 1;
- SEQ ID NO: 21 and SEQ ID NO: 22 for detecting mutation in SEQ ID NO: 2;
- SEQ ID NO: 25 and SEQ ID NO: 26 for detecting mutation in SEQ ID NO: 3;
- SEQ ID NO: 29 and SEQ ID NO: 30 for detecting mutation in SEQ ID NO: 4;
- SEQ ID NO: 33 and SEQ ID NO: 34 for detecting mutation in SEQ ID NO: 5;
- SEQ ID NO: 37 and SEQ ID NO: 38 for detecting mutation in SEQ ID NO: 6;
- SEQ ID NO: 41 and SEQ ID NO: 42 for detecting mutation in SEQ ID NO: 7;
- SEQ ID NO: 45 and SEQ ID NO: 46 for detecting mutation in SEQ ID NO: 8;
- SEQ ID NO: 49 and SEQ ID NO: 50 for detecting mutation in SEQ ID NO: 9;
- SEQ ID NO: 53 and SEQ ID NO: 54 for detecting mutation in SEQ ID NO: 10;
- SEQ ID NO: 57 and SEQ ID NO: 58 for detecting mutation in SEQ ID NO: 11;
- SEQ ID NO: 61 and SEQ ID NO: 62 for detecting mutation in SEQ ID NO: 12;
- SEQ ID NO: 65 and SEQ ID NO: 66 for detecting mutation in SEQ ID NO: 13;
- SEQ ID NO: 69 and SEQ ID NO: 70 for detecting mutation in SEQ ID NO: 14;
- SEQ ID NO: 73 and SEQ ID NO: 74 for detecting mutation in SEQ ID NO: 15;
- SEQ ID NO: 77 and SEQ ID NO: 78 for detecting mutation in SEQ ID NO: 16; or combinations thereof; wherein the presence of the substitution in the amplicon indicates the resistance to a specific antibiotic.
The method as claimed in claim 7, wherein the primer set comprises primers having a sequence selected from sequence as set forth in:
- SEQ ID NO: 19 and SEQ ID NO: 20 for detecting mutation in SEQ ID NO: 1;
- SEQ ID NO: 23 and SEQ ID NO: 24 for detecting mutation in SEQ ID NO: 2;
- SEQ ID NO: 27 and SEQ ID NO: 28 for detecting mutation in SEQ ID NO: 3;
- SEQ ID NO: 31 and SEQ ID NO: 32 for detecting mutation in SEQ ID NO: 4;
- SEQ ID NO: 35 and SEQ ID NO: 36 for detecting mutation in SEQ ID NO: 5;
- SEQ ID NO: 39 and SEQ ID NO: 40 for detecting mutation in SEQ ID NO: 6;
- SEQ ID NO: 43 and SEQ ID NO: 44 for detecting mutation in SEQ ID NO: 7;
- SEQ ID NO: 47 and SEQ ID NO: 48 for detecting mutation in SEQ ID NO: 8;
- SEQ ID NO: 51 and SEQ ID NO: 52 for detecting mutation in SEQ ID NO: 9;
- SEQ ID NO: 55 and SEQ ID NO: 56 for detecting mutation in SEQ ID NO: 10;
- SEQ ID NO: 59 and SEQ ID NO: 60 for detecting mutation in SEQ ID NO: 11;
- SEQ ID NO: 63 and SEQ ID NO: 64 for detecting mutation in SEQ ID NO: 12;
- SEQ ID NO: 67 and SEQ ID NO: 68 for detecting mutation in SEQ ID NO: 13;
- SEQ ID NO: 71 and SEQ ID NO: 72 for detecting mutation in SEQ ID NO: 14;
- SEQ ID NO: 75 and SEQ ID NO: 76 for detecting mutation in SEQ ID NO: 15;
-SEQ ID NO: 79 and SEQ ID NO: 80 for detecting mutation in SEQ ID NO: 16; or combinations thereof; wherein the presence of the substitution in the amplicon indicates the resistance to a specific antibiotic. The method as claimed in claim 6 or 7, wherein the sample is selected from the group consisting of blood, sputum, and spinal fluids. The method as claimed in claim 6 or 7, wherein the antibiotic is selected from pyrazinamide, rifampicin, ethambutol, streptomycin, or combinations thereof. A method of preparing a treatment regimen for a subject infected with M. tuberculosis, the method comprising the steps of: a. determining whether the subject infected with M. tuberculosis is resistant to an antibiotic using the method as claimed in claim 6 or 7; and b. selecting an antibiotic for which the subject having tuberculosis is not resistant as determined in step (a) for preparing a treatment regimen, wherein the antibiotic is selected from the group consisting of isoniazid, rifampicin/rifampin, ethambutol, pyrazinamide, rifabutin, rifapentine, streptomycin, moxifloxacin, ciprofloxacin, levofloxacin, ofloxacin, p- aminosalicylic acid, ethionamide, prothionamide, cycloserine, terizidone, capreomycin, kanamycin, amikacin, linezolid, delamanid, pretomanid, clofazimine and combinations thereof. A kit for determining resistance or susceptibility of a subject to a specific antibiotic for the treatment of tuberculosis, comprising one or more of primer set; wherein the primer set comprises primers having a sequence selected from sequence as set forth in:
- SEQ ID NO: 17 and SEQ ID NO: 18 for detecting mutation in SEQ ID NO: 1;
- SEQ ID NO: 21 and SEQ ID NO: 22 for detecting mutation in SEQ ID NO: 2;
- SEQ ID NO: 25 and SEQ ID NO: 26 for detecting mutation in SEQ ID NO: 3;
- SEQ ID NO: 29 and SEQ ID NO: 30 for detecting mutation in SEQ ID NO: 4;
- SEQ ID NO: 33 and SEQ ID NO: 34 for detecting mutation in SEQ ID NO: 5;
- SEQ ID NO: 37 and SEQ ID NO: 38 for detecting mutation in SEQ ID NO: 6;
- SEQ ID NO: 41 and SEQ ID NO: 42 for detecting mutation in SEQ ID NO: 7;
- SEQ ID NO: 45 and SEQ ID NO: 46 for detecting mutation in SEQ ID NO: 8;
- SEQ ID NO: 49 and SEQ ID NO: 50 for detecting mutation in SEQ ID NO: 9;
- SEQ ID NO: 53 and SEQ ID NO: 54 for detecting mutation in SEQ ID NO: 10;
- SEQ ID NO: 57 and SEQ ID NO: 58 for detecting mutation in SEQ ID NO: 11;
- SEQ ID NO: 61 and SEQ ID NO: 62 for detecting mutation in SEQ ID NO: 12;
- SEQ ID NO: 65 and SEQ ID NO: 66 for detecting mutation in SEQ ID NO: 13;
- SEQ ID NO: 69 and SEQ ID NO: 70 for detecting mutation in SEQ ID NO: 14;
- SEQ ID NO: 73 and SEQ ID NO: 74 for detecting mutation in SEQ ID NO: 15; or
- SEQ ID NO: 77 and SEQ ID NO: 78 for detecting mutation in SEQ ID NO: 16. The kit as claimed in claim 12, wherein the primer set comprises primers having sequence selected from sequence as set forth in:
- SEQ ID NO: 19 and SEQ ID NO: 20 for detecting mutation in SEQ ID NO: 1;
- SEQ ID NO: 23 and SEQ ID NO: 24 for detecting mutation in SEQ ID NO: 2;
- SEQ ID NO: 27 and SEQ ID NO: 28 for detecting mutation in SEQ ID NO: 3;
- SEQ ID NO: 31 and SEQ ID NO: 32 for detecting mutation in SEQ ID NO: 4;
- SEQ ID NO: 35 and SEQ ID NO: 36 for detecting mutation in SEQ ID NO: 5;
- SEQ ID NO: 39 and SEQ ID NO: 40 for detecting mutation in SEQ ID NO: 6;
- SEQ ID NO: 43 and SEQ ID NO: 44 for detecting mutation in SEQ ID NO: 7;
- SEQ ID NO: 47 and SEQ ID NO: 48 for detecting mutation in SEQ ID NO: 8;
- SEQ ID NO: 51 and SEQ ID NO: 52 for detecting mutation in SEQ ID NO: 9;
- SEQ ID NO: 55 and SEQ ID NO: 56 for detecting mutation in SEQ ID NO: 10;
- SEQ ID NO: 59 and SEQ ID NO: 60 for detecting mutation in SEQ ID NO: 11;
- SEQ ID NO: 63 and SEQ ID NO: 64 for detecting mutation in SEQ ID NO: 12;
- SEQ ID NO: 67 and SEQ ID NO: 68 for detecting mutation in SEQ ID NO: 13;
- SEQ ID NO: 71 and SEQ ID NO: 72 for detecting mutation in SEQ ID NO: 14;
- SEQ ID NO: 75 and SEQ ID NO: 76 for detecting mutation in SEQ ID NO: 15; or
- SEQ ID NO: 79 and SEQ ID NO: 80 for detecting mutation in SEQ ID NO: 16. The kit as claimed in claim 12, wherein the kit further comprises a DNA polymerase, extension nucleotides, and a buffer. A panel of nucleotide fragments for determining antibiotic resistance in a subject comprising at least one polynucleotide fragment of M. tuberculosis, having a nucleic acid sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15 or SEQ ID NO: 16, wherein:
- SEQ ID NO: 1 comprises a nucleotide substitution at C216T;
- SEQ ID NO: 2 has a nucleotide substitution at G705A;
- SEQ ID NO: 3 comprises a nucleotide substitution at A174C;
- SEQ ID NO: 4 comprises a nucleotide substitution at G1177C;
- SEQ ID NO: 5 comprises a nucleotide substitution at C674G;
- SEQ ID NO: 6 comprises a nucleotide substitution at C771T;
- SEQ ID NO: 7 comprises a nucleotide substitution at C343T;
- SEQ ID NO: 8 comprises a nucleotide substitution at G466T;
- SEQ ID NO: 9 comprises a nucleotide substitution at CCGA[2522]C;
- SEQ ID NO: 10 comprises a nucleotide substitution at T533G;
- SEQ ID NO: 11 comprises a nucleotide substitution at C112T;
- SEQ ID NO: 12 comprises a nucleotide substitution at G355A;
- SEQ ID NO: 13 comprises a nucleotide substitution at C383A;
- SEQ ID NO: 14 comprises a nucleotide substitution at T30C;
- SEQ ID NO: 15 comprises a nucleotide substitution at C643T; and
- SEQ ID NO: 16 comprises a nucleotide substitution at T633T; wherein, the presence of nucleotide substitution indicates resistance to antibiotic for treatment of tuberculosis.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106048019A (en) * | 2016-06-13 | 2016-10-26 | 遵义医学院附属医院 | Antituberculous drug drug-resistance gene and screening method thereof |
| IN201941006113A (en) * | 2019-02-15 | 2020-03-21 | ||
| IN202041055641A (en) * | 2020-12-21 | 2021-11-19 | ||
| US20210395798A1 (en) * | 2020-06-17 | 2021-12-23 | The Translational Genomics Research Institute | Early detection of drug-resistant mycobacterium tuberculosis |
| WO2022049365A1 (en) * | 2020-09-04 | 2022-03-10 | Quadram Institute Bioscience | Method and compositions for drug resistance screening |
-
2023
- 2023-05-18 WO PCT/IN2023/050468 patent/WO2023223354A1/en unknown
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106048019A (en) * | 2016-06-13 | 2016-10-26 | 遵义医学院附属医院 | Antituberculous drug drug-resistance gene and screening method thereof |
| IN201941006113A (en) * | 2019-02-15 | 2020-03-21 | ||
| US20210395798A1 (en) * | 2020-06-17 | 2021-12-23 | The Translational Genomics Research Institute | Early detection of drug-resistant mycobacterium tuberculosis |
| WO2022049365A1 (en) * | 2020-09-04 | 2022-03-10 | Quadram Institute Bioscience | Method and compositions for drug resistance screening |
| IN202041055641A (en) * | 2020-12-21 | 2021-11-19 |
Non-Patent Citations (3)
| Title |
|---|
| HE LEI, CUI PENG, SHI WANLIANG, LI QIONG, ZHANG WENHONG, LI MIN, ZHANG YING: "Pyrazinoic Acid Inhibits the Bifunctional Enzyme (Rv2783) in Mycobacterium tuberculosis by Competing with tmRNA", PATHOGENS, vol. 8, no. 4, 1 January 2019 (2019-01-01), pages 1 - 12, XP093113695, ISSN: 2076-0817, DOI: 10.3390/pathogens8040230 * |
| JENA LINGARAJA, NAYAK TAPASWINI, DESHMUKH SHRADDHA, WANKHADE GAURI, WAGHMARE PRANITA, HARINATH BHASKAR C: "Isoniazid with Multiple Mode of Action on Various Mycobacterial Enzymes Resulting in Drug Resistance", JOURNAL OF INFECTIOUS DISEASES & THERAPY, vol. 4, no. 5, 1 January 2016 (2016-01-01), pages 1 - 8, XP093113697, ISSN: 2332-0877, DOI: 10.4172/2332-0877.1000297 * |
| MORTIMER TATUM D, WEBER ALEXANDRA M, PEPPERELL CAITLIN S: "Signatures of Selection at Drug Resistance Loci in Mycobacterium tuberculosis", MSYSTEMS, vol. 3, no. 1, 1 January 2018 (2018-01-01), pages 1 - 16, XP093113698, ISSN: 2379-5077, DOI: 10.1128/mSystems.00108-17 * |
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